Recent groundbreaking discoveries have uncovered a role for lysine-specific histone methyltransferases in regulating eukaryotic gene transcription. One such enzyme in yeast, Set2p, modifies histone H3 on lysine 36 (H3K36) and this methylation event has been linked to the recruitment of histone deacetylase activity (RpdSS) that results in a more repressed chromatin environment. Set2p interacts with core components of the transcriptional machinery as well as other chromatin remodeling complexes. The full roles of Set2p in transcription and chromatin remodeling, however, have not been fully elucidated. We therefore propose to investigate the role of Set2p in these processes as well as mechanism by which Set2p, itself, is regulated. Recent evidence from the Strahl lab shows that Set2p is phosphorylated in vivo, and one site of phosphorylation has been found. Therefore, one goal is to fully map sites of phosphorylation within Set2p as well as define the kinases responsible. A related goal is subsequently to determine how Set2p affects downstream functions such as binding to RNA polymerase II or the recruitment of the Rpd3(S) deacetylase complex and what role, if any, Set2p phosphorylation plays in regulating these processes. A third goal will be to explore other functions of protein methylation by mining the yeast proteome for non-histone substrates of Set2p and other related methyltransferases. This goal has great potential to provide insight into the importance of protein methylation in biological processes. Collectively, these studies will make important contributions to our understanding of how histone methyltransferases regulate chromatin structure and function. Relevance to public health: Aberrant methyltransferase activity has been correlated with a number of human diseases including cancer. Understanding how methyltransferases modulate chromatin structure is essential to elucidating their role in these diseases. Therefore, this study has broad implications toward understanding both fundamental mechanisms of gene regulation and the role of posttranslational modifications in disease.